WFA causes vimentin to aggregate to a juxtanuclear location.

A) Immunofluorescence analysis of endogenous vimentin, tubulin and actin (white) in HEK293 cells treated for 4 h with 1–5 μM of WFA and compared to DMSO vehicle control. Cell nuclei were stained with DAPI (blue). Representative images are shown. Scale bar is equal to 10 μm. B) Immunoblot analysis of endogenous vimentin, tubulin and actin protein levels in HEK293 cells treated for up to 6 h with 1.5 μM of WFA, DMSO vehicle control and compared to untreated cells (Unt). Because vimentin (55 kDa), tubulin (50 kDa) and actin (42 kDa) resolve at similar sizes, equal amounts of cell lysate derived from the same experiment were run simultaneously on separate gels and immunoblots were processed in parallel. Densitometry values for vimentin, tubulin and actin are presented in (S2 Fig). C) The metabolic activities of HEK293 cells were assessed using concentrations of WFA between 100 nM—2 μΜ for up to 24 h and compared to untreated cells. Triton-X was used as a positive control of toxicity. D) i) HEK293 cells were untreated or treated with DMSO vehicle control or 1.5 μΜ WFA for 6h and stained with Annexin V/PI and analysed by flow cytometry. Two-way ANOVA ** p<0.01 (n = 2 independent experiments). ii) Cell morphology was analysed by microscopy prior to flow cytometry. Brightfield images were captured at 20-x magnification. Representative images are shown.